Synaptic vesicle (SV) exocytosis is mediated by SNARE proteins (v-SNAREs on the vesicle membrane, t-SNAREs on the target membrane), which form the core of a membrane fusion machine that is conserved among all eukaryotic cells. SV fusion results in the release of neurotransmitters into the synaptic cleft and thus mediates chemical communication between neurons. This process is tightly controlled by a number of accessory proteins, and this regulation is absolutely essential for temporal control of the cell-to-cell communication that underlies the function of the nervous system. In particular, the rapid and synchronous Ca2+- triggered fusion of SVs is controlled by the Ca2+-binding protein synaptotagmin 1 (syt1), but the mechanism of action of syt1 remains unclear. The overall goal of Aim 1 is to reconstitute full-length syt1 into functional v- SNARE proteoliposomes and to use this system to elucidate the mechanism by which these vesicles fuse with t-SNARE-bearing target membranes. Specific goals are to determine whether, and if so how, membrane- embedded syt1 contributes to the sequential docking, priming, and fusion of v- and t-SNARE vesicles. Innovations include the use of glass beads as templates to control, and thereby quantitatively explore, the role of membrane curvature - and the impact of syt1 on this parameter - during fusion. A further goal is to use fluorescent probes to monitor changes in the assembly of SNARE complexes during each step in the fusion pathway and to add-back a number of additional factors to better recapitulate fast and efficient, biologically relevant, membrane fusion in vitro. While syt1 acts as a major Ca2+ sensor for rapid evoked transmitter release, a second sensor mediates the delayed, asychronous component of evoked release. We have recently shown that at excitatory hippocampal synapses, the slow Ca2+ sensor, Doc2a, is required for asynchronous transmission. In Aim 2, we will take advantage of the homology between syt1 and Doc2 to generate a set of chimeras with graded changes in their kinetic properties. The resulting chimeras will be expressed in neurons that lack these proteins with the goal of precisely tuning the kinetics of synaptic transmission. In principle, it should be possible to make synapses with virtually any speed-of-response, and thereby determine the kinetic requirements for aspects of neural network function, including persistent reverberation and, in the long term, spike timing correlations. We will also extend our analysis of Doc2 function by using Doc2a and Doc2b KO and KD approaches to determine the role of these proteins in inhibitory synaptic transmission. Together, these experiments should make it possible to perturb network function for long term studies directed toward relating asynchronous release to aspects of memory, learning, and behavior.